Effect of Egg Washing and Correlation between Eggshell Characteristics and Egg Penetration by Various Salmonella Typhimurium Strains Vaibhav C. Gole 1 , Kapil K. Chousalkar 1 *, Juliet R. Roberts 2 , Margaret Sexton 3 , Damian May 4 , Jessica Tan 4 , Andreas Kiermeier 4 1 School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA, Australia, 2 School of Environmental & Rural Science, The University of New England, Armidale, NSW, Australia, 3 Primary Industries and Regions SA, Adelaide, SA, Australia, 4 South Australian Research and Development Institute, Adelaide, SA, Australia Abstract Salmonella is an important foodborne pathogen, causing an estimated 11,992 cases of infection in Australia per year. Egg or egg product related salmonellosis is a major concern for the egg industry. Worldwide, S. Typhimurium is one of the most common serovars identified in Salmonella food poisoning cases. The current study investigated the ability of five S. Typhimurium strains to penetrate washed and unwashed eggs using whole egg and agar egg penetration methods. All S. Typhimurium strains were able to penetrate eggshells and survive in egg albumen (at 20uC) according to whole egg penetration results. Polymerase Chain Reaction results demonstrated that S. Typhimurium strain 2 (10 3 and 10 5 CFU/mL), and strain 5 (10 3 and 10 5 CFU/mL) egg penetration was significantly higher (p,0.05) in washed eggs when compared to unwashed eggs. Statistical analysis of the agar penetration experiment indicated that S. Typhimurium was able to penetrate washed eggs at a significantly higher rate when compared to unwashed eggs (p,0.05). When compared to unwashed eggs, washed eggs also had significantly damaged cuticles. Statistical analysis also indicated that eggshell penetration by S. Typhimurium was related to various eggshell ultrastructural features such as cap quality, alignment, erosion, confluence, Type B bodies and cuticle cover. Citation: Gole VC, Chousalkar KK, Roberts JR, Sexton M, May D, et al. (2014) Effect of Egg Washing and Correlation between Eggshell Characteristics and Egg Penetration by Various Salmonella Typhimurium Strains. PLoS ONE 9(3): e90987. doi:10.1371/journal.pone.0090987 Editor: Axel Cloeckaert, Institut National de la Recherche Agronomique, France Received October 29, 2013; Accepted February 6, 2014; Published March 12, 2014 Copyright: ß 2014 Chousalkar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was conducted within the Poultry CRC, established and supported under the Australian Government’s Cooperative Research Centres Program. Salmonella isolates were obtained from Ms. Diane Davos, Australian Salmonella Reference Centre, Adelaide, South Australia, Australia. Mr. Vaibhav Gole is an International Postgraduate Research Scholarship recipient. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: *kapil.chousalkar@adelaide.edu.au Introduction Salmonella spp. have been one of the most important food poisoning pathogens throughout the last century and remain a challenge for microbiology and public health [1]. It is estimated that 1.3 billion incidences of nontyphoidal salmonellosis occur throughout the world annually [2]. The annual report of the OzFoodnet network [3] reported 11,992 cases of Salmonella infection in Australia in 2010, with an estimated annual cost due to all food borne illness of $1.2 billion [4]., Eggs are often implicated in the cases of food poisoning due to salmonellosis [3], which can be acquired by the ingestion of raw or undercooked eggs. Intact eggs can be contaminated by Salmonella using two possible routes: vertical transmission and horizontal transmission. Vertical transmission occurs as a result of Salmonella infection of the reproductive organs i.e. ovaries or oviduct hence also called the transovarian route. In the transovarian route, the egg yolk membranes or albumen surrounding are directly contaminated [5]. Horizontal transmission is also called the trans-shell route in which Salmonella penetrates through the eggshell during or following oviposition [6]. A major cause of egg related Salmonella food poisoning cases and the most prevalent serovar in the layer industry across the world is S. Enteritidis; however, it is not endemic in Australian layer flocks [7]. In Australia and other parts of the world, S. Typhimurium is one of the most common serovars identified in egg borne salmonellosis cases [3,36]. Horizontal transmission is the most common route by which salmonellae other than S. Enteritidis contaminate egg internal contents [8]. Important extrinsic factors such as the bacterial strain, temperature differential, moisture on the eggshell, the number of microorganisms in the inoculum and the storage conditions may affect eggshell penetration by Salmonella spp [5]. Intrinsic factors that may affect egg penetration include shell porosity, shell thickness and the extent of cuticle present on the shell [5]. There is also some evidence to suggest that eggshell translucency is associated with greater microbial penetration [9]. However, there is a lack of substantial literature on the relationship between translucency, eggshell ultrastructure and the penetration of bacteria. Faeces, water, caging material, nesting material, insects, hands, broken eggs, blood, soil or dust on the egg belt are the most common sources of microbial contamination of the eggshell [10,11]. Egg washing can reduce the microbial load on the PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e90987